1. Field of the Invention
The present invention relates to a method of using an optical apparatus for drug development and evaluation. More specifically, the present invention provides a convenient and cost effective method to evaluate the action of a drug at the cellular level, including its uptake, distribution, binding characteristics, etc.
2. Background Information
The determination of drug action at the cellular level is a problem of great importance to drug evaluation and development. Recently, the implementation of rational drug design, combinatorial chemistry techniques, and high throughput screening have led to large numbers of new potential drugs. However, currently there is no cost effective way to understand the details of how these potential drugs work at the cellular level. This lack of methodology requires pharmaceutical companies to spend millions of dollars in animal and clinical studies to evaluate a candidate drug.
The most direct way of evaluating a drug, however, is its actions at the cellular level. For example, the efficacy of a drug is generally determined by the following drug-cell interactions: (1) cellular distribution of the drug, (2) cellular uptake of the drug, (3) binding characteristics of the drug, and (4) biochemical pathways of the drug.
Another major obstacle of drug efficacy is the resistance of some cells to a drug. The underlying molecular and cellular mechanisms of this resistance are not totally understood. However, a number of mechanisms appear to contribute to the resistance: (1) increased efficiency of DNA repair mechanism after the DNA has been damaged by the drug, (2) decreased cellular uptake or increased efflux of drugs, (3) increased levels of “target” enzymes or alterations in “target” enzymes, (4) decreased drug efficacy because of increased drug breakdown, and (5) alternative biochemical pathways.
In addition to the efficacy of the drug, the safety of a drug must also be evaluated at the cellular level. For example, to identify if a drug has allow toxicity to normal cells but high toxicity to tumor cells generally requires an understanding of the unique biochemical differences between normal and abnormal cells.
Using methods in molecular biology to study drug actions at the cellular level is difficult if only conventional optical microscopes are used. This is because biomolecules are generally transparent in visible light and are therefore indistinguishable under optical microscopes. A molecularly selective imaging microscope (sometimes called a chemical imaging microscope) is needed to differentiate between molecular targets.
Laser scanning fluorescent microscopy, as a chemical imaging technique, has been routinely used for in vitro sample analysis for many years. Molecular imaging is acquired by choosing a stain or fluorescing agent that selectively, chemically or physically, bonds to specific regions of the sample. Quantitative measurements of intensity in fluorescence can provide images that illustrate the distribution of fluorescent markers in cells. The distribution of these markers determines the distribution of specific antibodies, ligand affinities, or covalent bonds that are tagged by the markers. However, the fluorescent approach has several disadvantages and limitations: (1) the sample preparation procedure is complicated and time consuming, (2) the fluorescent markers used in the specimen may cause undesireable pharmacological or toxicological effects, (3) suitable markers are not available for all biomolecules, (4) the fundamental problems of fluorophore photon bleaching during measurement severely limit the use of fluorescence microscopy, and (5) the relatively short wavelength used in fluorescence microscopy can easily cause photo-damage to the specimen.
Infrared microscopy is another chemical imaging technique that can provide molecular-specific images. An image of a sample is obtained by imaging the transmitted infrared radiation. Molecular selectivity is obtained by tuning the wavelength to a vibrational energy level of a selected molecular type in the sample. Since infrared imaging is derived from a material's intrinsic vibrational energy level, no external markers, dyes, or labels are required to contract the infrared image. However the spatial resolution of the image is usually several times the wavelength of infrared radiation. This is usually 10-20 μm, which is too large to resolve structures at the cellular level. In addition, many samples of biological interest are opaque in the infrared due to the presence of water since vibrational modes with a high change of dipole moment have a large infrared sensitivity. Consequently, it is often difficult, and sometimes impossible to obtain images of many molecular groups of interest by infrared microscopy.
Raman spectroscopy, in contrast to infrared techniques, is a technique for determining the vibrational modes of a molecule that is based on the scattering of a photon from the molecule. The Raman spectrum, formed by a plurality of scattered frequencies shifted from the illumination wavelength, has a long history of being used to distinguish different molecules. The Raman spectrum of a particular substance depends on the structure (vibrational states and chemical bonds) of the molecules. Therefore, a Raman spectrum can uniquely identify a particular type of molecule by its unique combination of scattered frequencies (also referred to as Raman peaks or Raman modes).
Raman images, acquired at selected Raman modes using a tunable filter, can provide an overview of the spacial arrangement of a particular type of molecule within a heterogenous specimen. Like infrared imaging, Raman imaging requires no external markers, dyes, or labels as required in fluorescent imaging. However, Raman scattering is superior to infrared absorption or transmission measurements of biological systems in that water has little effect on the Raman spectrum and, therefore, interference by water in Raman are negligible compared to infrared imaging. This is expected since the sensitivity of the vibrational mode in the Raman spectrum is related to the change in polarizability of the vibration, rather than a high change in dipole moment which is characteristic of infrared.
In addition, near-infrared excitation of biological systems has a number of advantages in Raman imaging. With this excitation source, Raman imaging produces less laser-induced fluorescence and photo-thermal degradation, and allows better perspective depth into a living cell.
Unfortunately, the signal for a Raman spectrum is inherently weak compared to the strength of the fluorescent signals, and therefore, can be difficult to detect. Consequently, Raman spectroscopy, especially Raman imaging, was not practical until the recent development of a number of new signal generation, processing and detection tools. Some examples are robust laser sources, holographic filters, and low-noise CCD (charge-coupled device) cameras. In addition, various Raman imaging techniques are being developed to enhance the Raman signal, for example, surface enhanced Raman imaging and coherent anti-stroke Raman imaging. The first commercial Raman imaging microscope became available in the early 1990s. Recently the Raman microscope has achieved resolution of 0.5 μm, and it is now feasible to obtain chemical imaging at the cellular level.
The present invention demonstrates that Raman imaging microscopy can be applied to the study of drug actions in a single cell. Specifically, the invention describes the methods of using Raman imaging microscopy to detect drug uptake, distribution, binding and metabolism in a single cell, and to study drug pharmacokinetics at the cellular level. Even though this application speaks to more conventional Raman imaging techniques, various enhanced Raman imaging techniques can be applied as well, including but not limited to, surface enhanced Raman imaging and anti-Stroke Raman imaging.